NIH-R15 ?Imaging Live Cells with Super-Resolution Microscopy? Project Summary Fluorescence optical microscopy is one of the most important tools available for the study of biological systems at the cellular level. Unfortunately, due to diffraction phenomena the resolution of fluorescence microscopes in the lateral dimension is limited to about 250 nm. As many biological structures within cells are much smaller than this, increasing resolution is of prime importance. Although several methods are now available which are able to extend the resolution of optical microscopes beyond the diffraction limit, imaging live cells with these methods remains a major challenge. Super-resolution structured illumination microscopy (SIM), which can achieve a resolution of approximately 100 nm, is a suitable super-resolution method for imaging live cells. However, adoption of this technique by biologists is hindered by the inflexible equipment and artifact-prone image analysis algorithms which are currently available. The solution to this problem demands innovations in both optical design and in data processing methods which are used in SIM. Another new method, single molecule localization microscopy, achieves much higher resolution. Although live cell imaging has been demonstrated using single molecule localization approaches, applicability of this method in live cell studies is extremely limited due to the need to collect several thousand images to reconstruct a single super-resolution image. One solution to this problem is to greatly increase the density of photoactivated molecules in a given camera frame, but doing so requires more sophisticated computational methods to produce a satisfactory result. The goal of this interdisciplinary project is to develop, improve, and utilize super-resolution microscopy with a focus on imaging live cells. In Aim 1 we will develop alternative illumination approaches for SIM using economical components, and we will develop and implement improved SIM reconstruction algorithms which produce higher quality, more reliable results than are available with current methods. In Aim 2, we will improve single molecule localization microscopy, by developing and implementing algorithms for analysis of images with high densities of photoactivated molecules. Using the high density single molecule methods we develop, we expect to be able to accelerate imaging speed by a factor of 50, with a resulting image resolution of approximately 20 nm. In Aim 3, we will use the newly developed methods for studies of the molecular basis of allergic responses. We will use a novel total internal reflection-SIM microscope with polarized excitation to reveal the relationship between cell surface receptors and the morphology of the plasma membrane, in particular membrane regions with high curvature.